A high-throughput technology, termed DMS2 (DMS square) for Direct Microarray-based Screening of Differentially Methylated CpG Sites, has been developed for the discovery of abnormally methylated sites as cancer molecular markers. Combining the methylation specificity of restriction enzymes, the sensitivity of the polymerase chain reaction, and the high processibility of microarray hybridization, DMS2 has the potential of rapidly interrogating tens of thousands of genomic loci for abnormal methylation patterns associated with cancers. The technology is designed to be sensitive to the full range of possible methylation changes found in many cancers. It is expected that the technology will allow the testing of hundreds of samples a day in a typical molecular biology laboratory, making it an efficient discovery and diagnostic tool for cancer and other diseases. At the Phase I stage of the SBIR, the technology will be further validated through the building of a methylation microarray representing 100 selected cancer-related genes; the assessment of sensitivity, specificity and high throughput compatibility; and the application of DMS2 technology and the microarray in analyzing methylation changes in breast cancer progression. PROPOSED COMMERCIAL APPLICATION: High throughput methylation analysis technology will lead to the discovery of cancer-specific molecular markers and will also be used for efficient profiling of clinical samples for not only diagnosis, but also patient stratification, drug therapy monitoring, and toxicology. Through early diagnosis of cancers, including breast, lung, colorectal, and prostate cancers, the value of the technology will be enormous.